DE4212706A1 - Diagnostic method for the immunological determination of one or more analytes - Google Patents

Abstract

The invention relates to an improved diagnostic method for the immunological determination of one or more analytes by means of specific binding partners, where one specific binding partner is immobilised on a carrier and the extent of the binding of the analyte to the first specific binding partner is determined by means of a second binding partner which is specific for the analyte and is labelled either directly or via other binding partners.

Description

The invention relates to an improved diagnostic Method for the immunological determination of a several analytes using specific binding partners, the one specific binding partner to one The carrier is immobilized and the extent of binding of the Analytes to the first specific binding partner by means of a second specific for the analyte Binding partner, either directly or through others Binding partner is marked, is determined.

The "Neural Cell Adhesion Molecule" (NCAM) is an inter cellular adhesion protein that occurs in various, pre all neural tissues of humans but also of animals, such as B. mice, chickens etc. is expressed. There were three isoforms with different molecular weights described, which also additionally, for. B. by under different glycosylation patterns can be further modified (Nybroe et al., Neurochem. Int., 12: 215-262, 1988). NCAM is also from immunohistochemical studies as a tumor-associated human antigen, small small cell lung cancer (SCLC) (Kibbelaar et al., J. Pathol. 159: 23-28, 1989). With an immunoassay based on the use of the for α-2,8 linked N-acetyl-neuraminic acid chains (2,8-NAcN) specific MAK 735 (Frosch et al., Proc Natl. Acad. Sci. 82: 1194-1198, 1985; Finne et al., J. Immunol. 138: 4402-4407, 1987) as a solid phase antibody  in combination with the MAK BW SCLC-1 (ECACC deposit no. 90 022 110) or BW SCLC-2 (ECACC Deposit No. 90 022 109) recognized the so-called "embryonic" NCAM isoform shown that this isoform is surprisingly or a de Finished antigen (T-NCAM) in the serum of a large number of SCLC patients occurs in increased concentration (EP-A-0 443 599 A2).

Various are used for pregnancy monitoring Diagnostic procedures are used to get fetal early To be able to discover malformations. Has proven itself u. a. the immunochemical determination of alpha-fetoprotein (AFP) in amniotic fluid or maternal serum. Increased AFP concentrations indicate a neural tube defective in the embryo.

Due to false positive and false negative results however, additional is recommended for this indication To conduct investigations. Through the work of Ibsen et al. (J. Neurochem. 41: 363-366, 1983) is still known that an increased amniotic fluid concentration of Neural cell adhesion molecules (= D2) an indication of fetal malformations.

The work of Ibsen et al. is however also to ent that the diagnostic selectivity of your D2- Testes, d. H. the possibility between healthy and patho to be able to distinguish logical samples with mothers serum as sample material that is required for a first Testing as part of pregnancy monitoring is more accessible, is insufficient.

In addition, it was found that when the T- NCAM tests from a microtitration plate version a tube version of the T-NCAM concentration in sera healthy blood donors were found higher during the  T-NCAM concentration in sera from tumor patients in was found to be the same in both tests. The cause an NCAM isoform contained in the samples can be used for this be with other polysialic acid chains that with the Solid phase antibodies as well as the tracer / conjugate antibodies cross-reacted and those with the immunochemical somewhat reak is able to bind more solid tube phase better.

The object of the present invention was therefore to improved T-NCAM test, preferably for the tumor diagnostics and pregnancy monitoring, to develop where the diagnostic selectivity, d. H. the Differentiation into pathological and normal samples, is improved.

Surprisingly, it was found that the addition suitable amounts of substances (such as the Capsule polysaccharide from E. coli K 1) α-2,8 linked Contain N-acetyl-neuraminic acid chains and specifically from the "solid phase antibody" - e.g. B. in the T-NCAM test used MAK 735 - but not from the marked ones "Tracer / Conjugate Antibodies" - e.g. B. in the T-NCAM test used MAK BW SCLC-1 or -2 - bound to Sample incubation buffers solve this problem could.

The invention thus relates to a method for immunological determination of one or more analytes by means of specific binding partners, the one specific binding partners immobilized on a carrier and the degree of binding of the analyte to the first specific binding partner by means of a second for the analyte-specific binding partner, which either is marked directly or via other binding partners, is determined, the reaction with at least one the specific binding partner in the presence of a Suppressor substance takes place, which has a high affinity to  this specific attachment partner, but not for the another or one of the other specific binding partner.

Such a method is preferred, in which the first specific binding partners specifically α-2,8 linked N-acetyl-neuraminic acid chains can bind and the second specific binding partner for the same epitope is specific, like the monoclonal antibodies BW SCLC-1 and BW SCLC-2.

The invention further relates to such a method ren, the binding partner to which the suppressor sub punch has an affinity to which the solid phase is bound.

The suppressor substance used contains before preferably α-2,8 linked N-acetyl-neuraminic acid Chains.

The suppressor substance is particularly preferably from components of the capsule polysaccharide from E. coli K 1 or meningococcal type B or from Colominic Acid, whole preferably from Colominic Acid.

The suppressor substance is in such a concentration used that the measurement signal of the test standard is not around more than 50% is inhibited, preferably by 20 to 40%, most preferably around 30%.

In such a process, Colominic Acid is used in a Concentration of 0.01 to 10 µg / ml used, preferred usually from 0.1 to 1 µg / ml.

One possible mode of action of these substances is following: the 2,8-NAcN that are not approved by the tracer / Conjugate antibodies are recognized, compete  presumably with the T-NCAM molecules and the "cross reacting substance "around the free binding sites of the MAK 735. Due to the different binding of T-NCAM and 2,8-NAcN, e.g. B. on the MAK 735, less Molecules of the "cross-reacting substance" that a ge have less affinity for the solid phase antibody than T-NCAM or 2,8-NAcN, bound and thus will be final the diagnostic selectivity of the T-NCAM test significantly improved. Crucial for choosing 2,8-NAcN is their binding ability with the solid phase anti body. Other glycoproteins that are not specific to MAK 735 are bound, but increased sialic acid have content such. B. Fetuin (Sigma), Mucine (Type I-S, Sigma), α1-acid glycoprotein (Behringwerke), or Sialic acids (e.g. Type VI or Type VIII, Sigma) remained ineffective even at a concentration of up to 0.1 mg / ml.

The method found for the T-NCAM test can be found in similarly used for other tests, e.g. B. for Antibody-antigen-antibody "sandwich" immunoassays or for antigen-antibody-antigen "sandwich" immuno assays.

This involves binding one contained in the samples cross-reactive substance used by both in the test set specific receptors R1 and R2 is recognized, to one of the specific receptors R1, preferably that Solid phase bound, thereby preventing the Pro beninkubationsmedium a suppressor substance in suitable ter concentration is added, which thereby characterized is that it competitively binds the cross reactive substance inhibited at R1 and no specific Has binding site for the receptor R2.

A similar improvement as with the very small amounts of a specific suppressor substance could only be achieved with much higher concentrations of salts such as NaCl or CaCl ₂ . Good results were achieved when using a sample incubation buffer with a NaCl concentration of more than 150 to 2000 mmol / l, especially in the range from 250 to 1000 mmol / l. Carbonate buffer with a pH <8.0 (particularly good pH 8-10) and TRIS or phosphate buffer in the range of pH 6-8 have proven to be particularly suitable as buffers.

The diagno The static sensitivity of the T-NCAM test increased significantly be tied.

The following examples serve to explain the Erfin and do not restrict them in any way.

example 1 Enzyme immunoassay for T-NCAM determination in human bodies liquids

To determine the concentration of T-NCAM, each 10 µl sample material and 100 µl sample buffer (OSND, Behringwerke) in the MAK 735 after one subject known processes coated wells of Microtitration plates (Nunc) pipetted and one hour incubated at 37 ° C.

After washing three times with the diluted Enzygnost Wash buffers (OSEW, Behringwerke) were in the individual Wells each 100 µl of the MAK BW SCLC-1-POD or BW SCLC-2-POD conjugates filled in (POD = horseradish oxidase). The following one-hour incubation step at + 37 ° C was abge with a three wash cycle closed.

For the third incubation step at room temperature, 100 µl of a buffer / substrate chromogen solution (H ₂ O ₂ / TMB; OUVG / OUVF, BW) were then pipetted into the wells and the enzyme reaction after 30 minutes with Enzygnost stop solution (OSFA , BW) ended. The extinction measurement of the samples was carried out at 450 nm. The T-NCAM concentrations of the samples could be determined quantitatively using a standard series (unit: U / ml).

Example 2 Diagnostic procedure for pregnancy monitoring

The concentration of T-NCAM in amniotic fluid samples was determined analogous to the immunochemical described in Example 1 Procedure determined. The AFP concentration was measured with a commercially available test.

Result: In amniotic fluid samples was present embryonic malformations (e.g. spina bifida or anence phalie) increased T-NCAM concentrations measured (Table 1: Samples 52, 56 u. 159). With samples 53 and 139 with pathologically increased AFP concentration is the NCAM Concentration according to the clinical finding of a healthy embryos in the normal range. Out of this must to be concluded that there is a diagnostic procedure to determine T-NCAM or NCAM for pregnant women surveillance (early detection of embryonic malformations dung).  

Table 1:

T-NCAM concentration in amniotic fluid samples

Example 3 Chemiluminescence immunoassay for T-NCAM determination in human body fluids

To determine the concentration of T-NCAM, each 20 µl sample material and 200 µl sample buffer (e.g. OSND, Behringwerke) in the MAK 735 according to the Processes known to those skilled in the art coated polystyrene pipetted (Greiner) and an hour at room temperature and incubated with shaking.

After washing twice with 2 ml BeriLux wash buffer (Behringwerke) were each in the individual wells 200 µl of a solution of the with acridinium-N-acylsulfonamide labeled MAK BW SCLC-1 or BW SCLC-2.  

The following one-hour incubation step at room temperature and with shaking was done with a three wash cycle completed.

Subsequently, each was automatically in a luminometer 300 µl BeriLux analyzer reagent R1 and R2 (Behringwerke) filled into the tubes and the luminogenic measurement signal measured over a period of 3 seconds. Using one the T-NCAM-Konzentra tions of the samples are determined quantitatively (measure unit: U / ml).

Example 4 Add 2.8-NAcN to the sample incubation buffer for Ver improvement of normal sera / tumor sera discrimination

The T-NCAM concentration of sera from healthy blood donors or of samples containing T-NCAM, e.g. B. of patients with small cell lung cancer, have been identified with the immunche mix test method determined according to Example 3. Here the sample incubation medium (OSND, Behringwerke) mixed with various substances, including Connections such as B. with a preparation of the capsule po lysaccharides from E. coli K 1 (see also Rohr & Troy, J. Biol Chem. 255: 2332-2342, 1980; Weisgerber & Troy, J. Biol Chem. 265: 1578-1587, 1990) or Colominic Acid (Sigma)), such as B. also the capsule polysaccharide of Meningo cocci type B α-2,8 linked N-acetyl-neuraminic acid Wear chains and specifically from MAK 735, but not from the MAK BW-SCLC-1 or -2 are bound. The admitted Amount of 2,8-NAcN was selected so that the measurement signal the test standards were inhibited between 1 and 50%. Other glycoproteins with an increased sialic acid gene stop, such as B. Fetuin (Sigma), Mucine (Type 1-S, Sigma), α1-acid glycoprotein (Behringwerke), or sialic acids (e.g. Type VI or Type VIII, Sigma) up to one Concentration of 0.1 mg / ml used.  

Result

Only by adding 2,8-NAcN could one improved tumor sera / normal sera discrimination can be achieved (see example in Table 2 and figure).

Table 2:

T-NCAM determination in standard series and tumor sera using a 2.8-NAcN-containing progen buffer

(Addition of E. coli K 1 capsule polysaccharide)

Example 5 Addition of salts / ions to the sample incubation buffer Improvement of normal sera / tumor sera discrimination

The T-NCAM concentration of sera from healthy blood donors or of samples containing T-NCAM, e.g. B. of patients with small cell lung cancer, have been identified with the immunche mix test method determined according to Example 3. Here was a 50 mM Tris / HCl buffer, pH 7.0, which further Contained 0.1% human serum albumin and 0.5% Tween 20 mixed with various salts and as a sample incubation buffer used (results see table 3).  

Table 3:

T-NCAM determination in normal sera and samples containing T-NCAM

Description of the figure Figure

T-NCAM serum concentration of healthy blood donors and patients with small cell lung cancer (SCLC) determined using a T-NCAM test a sample incubation medium (OSND, Behringwerke) with and without (control) addition of 0.5 µg / ml Colominic Acid (Sigma).

Claims (7)

1. A method for the immunological determination of one or more analytes by means of specific binding partners, the one specific binding partner being immobilized on a support and the extent of binding of the analyte to the first specific binding partner by means of a second binding partner specific for the analyte, which is either direct or is marked via further binding partners, is true, characterized in that the reaction takes place with at least one of the specific binding partners in the presence of a suppressor substance which has a high affinity for this specific binding partner, but not for the other or one of the further specific binding partners having. 2. The method according to claim 1, characterized in that the first specific binding partner specifically α-2,8-linked N-acetyl-neuraminic acid chains to bin the ability and the second specific attachment partner is specific to the same epitope as the monoclo nalen antibodies BW SCLC-1 and BW SCLC-2. 3. The method according to claim 1 or 2, characterized net that the binding partner to which the suppressor substance has an affinity, bound to the solid phase is. 4. The method according to claim 1, 2 or 3, characterized records that the suppressor substance is α-2.8 linked Contains N-acetyl-neuraminic acid chains. 5. The method according to claim 1, 2 or 3, characterized records that the suppressor substance from components of capsule polysaccharide from E. coli K 1 or Meningo  cocci type B or from Colominic Acid, preferably consists of Colominic Acid. 6. The method according to claim 1, 2 or 3, characterized records that the suppressor substance in such Concentration is used that the measurement signal of Test standards is inhibited by no more than 50%. 7. The method according to claim 1, 2 or 3, characterized records that Colominic Acid in a concentration from 0.01 to 10 µg / ml is used.